Biofeedback electrostimulation for bionic and long-lasting neural modulation

Invasive electrical stimulation (iES) is prone to cause neural stimulus-inertia owing to its excessive accumulation of exogenous charges, thereby resulting in many side effects and even failure of nerve regeneration and functional recovery. Here, a wearable neural iES system is well designed and built for bionic and long-lasting neural modulation. It can automatically yield biomimetic pulsed electrical signals under the driven of respiratory motion. These electrical signals are full of unique physiological synchronization can give biofeedback to respiratory behaviors, self-adjusting with different physiological states of the living body, and thus realizing a dynamic and biological self-matched modulation of voltage-gated calcium channels on the cell membrane. Abundant cellular and animal experimental evidence confirm an effective elimination of neural stimulus-inertia by these bioelectrical signals. An unprecedented nerve regeneration and motor functional reconstruction are achieved in long-segmental peripheral nerve defects, which is equal to the gold standard of nerve repair -- autograft. The wearable neural iES system provides an advanced platform to overcome the common neural stimulus-inertia and gives a broad avenue for personalized iES therapy of nerve injury and neurodegenerative diseases.

The manufacturing process of CS/PEDOT/PCL conduit (MF-NGC). The PCL solution (10wt% in dichloromethane) was sprayed on the surface of the CS/PEDOT nanofibrous conduit using the microsyringe pump to obtain the resulting MF-NGC.

Note 3. Characterizations of MF-NGC.
The tensile tester (UTM2303) was employed to measure the mechanical properties of MF-NGC. The conductivity of MF-NGC was measured by the four-probe method via M3 four-probe tester (Suzhou Jingge Electronics Co., Ltd.) and then calculated according to the following formula 2 : Where W is the thickness of the sample, S represents the probe distance and D(d/s) represents the correction value, which is obtained by looking up the table based on the length of the long side and the short side of the sample.
The CV curve of the MF-NGC was measured by an electrochemical workstation (Chi 760e) and a three-electrode system. The MF-NGC were immersed in an SBF solution at 37 ℃ to simulate the degradation behavior in vivo and the mass loss of MF-NGC under dry condition were measured by a high-precision balance (BSA224S, Sartorius Scientific Instruments Co., Ltd.).
Note 4. The synchronism measurement between Bio-iES and respiratory movement SD rats were anesthetized by the intake of isoflurane gas and then fixed on the operating table. The middle neck of SD rats was cut to expose the trachea, and a breathing tube was intubated into the trachea. The breathing tube was connected to a tension transducer to record the tidal volume breathing curve. Meanwhile, the phrenic nerve and vagus nerve were separated to be contacted with hook-working electrodes.
The grounding electrode was connected to the rats' skin. All signals were input to the multi-channel physiological signal acquisition and recording instrument (RM6240).
The electronic bandage was attached to the abdomen of rats and the waveform of Bio-iES was displayed by a digital oscilloscope (HMO3002). 20 minutes after surgery, the breathing curve, phrenic and vagus nerve discharge curve, and Bio-iES signals were recorded, simultaneously. Meanwhile, the electrochemical workstation provided the square-wave (Sw-iES group) and triangular-wave (Tw-iES group) electrical stimulation as control groups

Note 5. The growth and development on nerve cells under Bio-iES
(1.5V, 1.5Hz). Tuj1/MBP/DAPI triple fluorescence staining was used to quantitatively analyze relative neuronin expression level after electrical stimulation, and the neurite length and relative specific protein levels were measured by Image J analysis software.
Measurement of cell membrane potential. Motor neuron cultured on conductive mat were continuously stimulated by Bio-iES, Tw-iES and Sw-iES for 48h, respectively.
Then, the membrane potential was measured using a whole-cell patch-clamp technique.
Before measurement, the cells were washed three times and then bathed in extracellular recording solution. Electrophysiology data were recorded by MultiClamp 700B (Axon Instruments) system. The evoked membrane voltage response was stimulated by injecting current in current-clamp mode (200 pA, 270 ms).

Note 6. Implantation of MF-NGC in vivo.
Sixty SD rats (male, 150-200g, 6-7 weeks) were randomly selected for in vivo experiments. They were evenly divided into fout groups: MF-NGC with square wave electrical stimulation named as Sw-iES group; MF-NGC with triangular-wave electrical stimulation named as Tw-iES group; MF-NGC with electronic bandage named as Bio-iES group. The autograft group was set as a control group by rotating 180° of severed nerve and being re-implanted into the defect area. For all four groups, rats were anesthetized by the intake of isoflurane gas (0.8-1.5%) and maintained with 1.0% isoflurane. Simultaneously, a 15 mm defect of the sciatic nerve was served as a nerve injury model.

Note 7. Long-term biocompatibility of MF-NGC in vivo.
The long-term biocompatibility of MF-NGC was assessed by CD68 and TNF-α immunofluorescent staining, respectively. After postoperation within two months, the sciatic nerve samples were taken out to assess the inflammatory caused by the implantation of MF-NGC. All the samples (1 st week, 4 th week, 8 th week) were dehydrated, then embedded in paraffin, sliced into slices by using a microtome. The CD68 and TNF-αimmunofluorescence were employed to assess the inflammation level in the sciatic nerve. The observed image was obtained via an immunofluorescence microscope (Leica).

Note 8. Histological assessment of the regenerated nerve.
Morphology assessment of the regenerated nerve. The regenerated nerve was dissected immediately after the electrophysiological evaluation. HE staining, TB staining, and TEM analysis were used to observe the cross-sectional morphology of regenerated nerves, respectively. For HE staining, all nerve samples were fixed with 4% paraformaldehyde, then embedded in paraffin, cut into sections by using a microtome.
For TB staining, and TEM, all nerve samples were fixed with 2.5% glutaraldehyde, then embedded in Epon812 resin, cut into ultrathin sections. The HE staining and TB staining were observed by using an immunofluorescence microscope (Leica). The superfine microstructure of regenerated myelin sheath was observed via a TEM (China Titan) at a voltage of 80 kV. The diameter of the myelin sheath, the diameter of the myelin axon, the thickness of the myelin sheath were measured by Image J software.
Histological assessment of neovascularization on the regenerated nerve. At 12 weeks postoperatively, the regenerated sections of sciatic nerves were used for CD34 and VEGF immunofluorescence and CD31immunohistochemical assays as described above. The primary antibodies (Abcam, USA) included, anti-CD34 (1:400) and anti-CD31 (1:150), anti-VEGF (1:500 Abcam, USA). The microvessel density (MVD) and CD 31 area were measured by Image J software.
Histological assessment of typical calcium-dependent signaling on the regenerated nerve. At 12 weeks postoperatively, the regenerated sections of sciatic nerves were used for c-fos and BDNF immunofluorescence assays as described above.

Note 9. Functional recovery analysis.
We performed a walking trajectory analysis to measure functional recovery. SFI values were calculated by the formula reported by previous study. 3 Fig. 12 The bioelectronic bandage was fixed on the abdomen of SD rats to be derived by the respiratory movement for accelerating the restoration of PNI.
A breathable clothes was worn on the SD rat to avoid slide displacement of bioelectronic bandage during the daily activity. Supplementary Fig. 15 (a) The walking footprint of Sw-iES group, Tw-iES, Bio-iES, autograft, and normal groups at 12 weeks that acquired by the camera. (b) The SFI value was calculated from the walking footprint. (n = 5,  : p < 0.001).